human cd63 mouse dshb h5c6  (Developmental Studies Hybridoma Bank)

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: DsRed-CD63 and CD9 localization in Rab5 endosomes in MCF7 cells. (A) MCF7 cells were transfected with each siRNA as indicated, then transfected with GFP-Rab5Q79L (green) and DsRed-CD63 (red), fixed and stained with anti-CD9 antibody (blue). The representative images of perinuclear region with enlarged endosomes are shown. Rab5 endosomes are indicated with arrowheads. Note that Rab5 endosomes 100% filled with CD63 also contained CD9 partially or 100%. In ADAP1 and ARAP1KD cells, Rab5 endosomes lost CD63 and CD9 signal. (B) The experiment in A was repeated three times. More than 10 cells were counted per experiment and more than 30 cells were classified as indicated. The percentages of each cell were shown with s.d. The culminated means of each fraction are shown on top of each bar. Control, n= 40; ADAP1KD, n= 30; ARAP1KD, n= 32. (C) The same data in B were used to make the graph of transposed x axis and classification as indicated. Scale bars: 5 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Staining, Control

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: In ADAP1 and ARAP1KD cells, CD63 incorporation in endosomes is inhibited. (A) MCF7 cells were transfected with control and ADAP1 siRNAs, and the cell lysates were subjected to western blotting. Note that ADAP1 was efficiently downregulated in ADAP1 siRNA-transfected cells compared with control siRNA-transfected cells. (B) A similar experiment for ARAP1. Note that ARAP1 was efficiently downregulated by its siRNA. (C) The siRNA-transfected cells were transfected with GFP-Rab5Q79L (green) and stained with anti-CD63 (red). The insets were enlarged and Rab5 positive endosomes are indicated with arrowheads. Note that CD63 was filled in endosomes in control cells, whereas CD63 signal was lost in ADAP1 and ARAP1KD cells. (D) The Rab5-positive endosomes were classified 100% filled, partially filled, and none with CD63. The percentage of Rab5 endosomes in each category per cell was shown. More than 15 cells were counted per experiment and the experiment was repeated four times. Error bar, standard deviation (s.d.). Control, n= 91; ADAP1KD, n =73; ARAP1KD, n= 67. Significance was calculated by two-way ANOVA with Sidak's multiple comparisons test. ns, not significant, **** P< 0.0001. Scale bars: 10 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Control, Western Blot, Staining, Standard Deviation

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: The increase in endosomes without CD63 in ADAP1 and ARAP1KD cells is not due to off-target effects. (A) MCF7 transfected control or ADAP1 siRNA were transfected with human ADAP1-HA (ADAP1 overexpression; ADAP1OE, blue) and GFP-Rab5Q79L (green). CD63 was stained with the CD63 antibody (red). The insets were enlarged and Rab5 endosomes are indicated with arrowheads. CD63 signal was seen in the control, while the signal disappeared in ADAP1KD cells. In ADAP1KD+ADAP1OE cells, the CD63 signal in the endosome was recovered. ADAP1OE cells did not show much effect on CD63 in endosomes. (B) The experiment in A was repeated three times. More than 10 cells were counted per experiment and total more than 30 cells were quantified. The percentage of endosomes without CD63 was shown with s.d. Control, n= 33; ADAP1KD, n= 34; ADAP1KD+ADAP1OE, n= 37; ADAP1OE, n= 31. The Mann–Whitney test was performed. **** P< 0.0001; ns, not significant. (C) MCF7 was transfected with siRNA ARAP1 number 2 as indicated and subjected to western blotting. (D) The cells were transfected with control or ARAP1 number 2 siRNA and immunofluorescence was performed as in A. (E) The experiment in D was repeated three times. More than 10 cells were counted per experiment and total more than 30 cells were quantified. Rab5 endosomes without CD63 was shown as in B. Control, n =35; ARAP1 number 2, n =31. The Mann–Whitney test was performed, ** P< 0.01. (F) ARAP1 number 3 siRNA was tested for western blotting as in C. (G) Immunofluorescence was performed for control and ARAP1 number 3 cells. (H) The experiment in G was repeated three times. More than 10 cells were counted per experiment and total more than 30 cells were quantified. Rab5 endosomes without CD63 were quantified. Control, n =34; ARAP1 number 3; n =37. The Mann–Whitney test was performed, **** P< 0.0001. Scale bars: 10 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Control, Over Expression, Staining, MANN-WHITNEY, Western Blot, Immunofluorescence

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: EGF and CD63 localization in Rab5 endosomes in HeLa cells. (A) HeLa cells were transfected with each siRNA as indicated, then transfected with GFP-Rab5Q79L (green), and internalized EGF-Alexa 555 for 40 min (red), fixed and stained with anti-CD63 antibody (blue). The representative images of perinuclear region with enlarged endosomes are shown. Rab5 endosomes are indicated with arrowheads. Note that Rab5-endosomes contained dotty signal of EGF, but not CD63. (B) The experiment in A was repeated three times. More than 10 cells were counted per experiment and more than 30 cells were classified as indicated. The percentages of each cell were shown with s.d. The culminated means of each fraction were shown on top of each bar. Control, n= 32; ADAP1KD, n= 32; ARAP1KD, n= 34. (C) The same data set in B were used for the graph of transposed x axis and classification as indicated. Scale bars: 5 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Staining, Control

Journal: Cell reports

Article Title: Recruitment of DNA to tumor-derived microvesicles

doi: 10.1016/j.celrep.2022.110443

Figure Lengend Snippet: (A) LOX melanoma cells were fixed and stained to examine the intracellular localization of ARF6 (green) and CD63 (blue). CD63 is not detected at the cell periphery in nascent TMVs (inset). (B) Intracellular pools of ARF6 (red) and CD63 (green) were imaged by confocal microscopy and colocalization measured using Pearson’s R (n = 20 cells from n = 3 biological replicates). (C) LOX cells were fixed and stained as indicated. Confocal microscopy reveals the inclusion of dsDNA, together with ARF6, within budding TMVs. (D) TMVs and exosomes were isolated from conditioned media as described previously. Equal amounts of protein lysates from whole cells, TMVs, and exosomes were then separated by SDS-PAGE and histone H3 content was examined by western blotting. (E) EVs isolated from iodixanol gradient fractions were lysed, separated by SDS-PAGE, and histone H3 and trimethyl H3K9 content examined by western blotting. (F) Histone H3 and dsDNA contents were examined by confocal microscopy in shedding melanoma cells. Histone near the nucleus co-labels with dsDNA (arrowheads), while punctate histone H3 nearer the cell periphery does not (arrows). (G) Histone H3 and dsDNA contents were examined in nascent TMVs by immunofluorescence. Only a small pool of TMV-associated histone co-labels for dsDNA (arrowhead inset). (H) Amphisome markers histone H3 and CD63 content were examined by immunofluorescence. Similar to previously published reports, histone H3 and CD63 can be identified juxtaposed where they are likely to reside within the same late endosomal structure (arrowheads). (I) Orthogonal view of histone H3 and CD63 immunofluorescent image reveals co-trafficking of histone H3 and CD63 to the cell periphery where they are released without containment within TMVs. Scale bars, 10 μm.

Article Snippet: Mouse monoclonal antibody against human CD63 (H5C6) used for immunofluorescence was purchased from the Developmental Studies Hybridoma Bank.

Techniques: Staining, Confocal Microscopy, Isolation, SDS Page, Western Blot, Immunofluorescence

Journal: Cell reports

Article Title: Recruitment of DNA to tumor-derived microvesicles

doi: 10.1016/j.celrep.2022.110443

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal antibody against human CD63 (H5C6) used for immunofluorescence was purchased from the Developmental Studies Hybridoma Bank.

Techniques: Virus, Control, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, Imaging, dsDNA Assay, Plasmid Preparation, Software, Magnetic Beads, Transfection, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Isolation of Human Small Extracellular Vesicles and Tracking of Their Uptake by Retinal Pigment Epithelial Cells In Vitro

doi: 10.3390/ijms21113799

Figure Lengend Snippet: Isolation, characterization, and labelling of the urinary small extracellular vesicles (EVs). ( A ) TEM images of the small EVs obtained using ultrafiltration (UF). UF samples showed intact cup-shaped double-membrane structures. Scale bar = 0.5 µm. ( B ) Nanoparticle tracking analysis (NTA) showing the size distribution of the urinary small EVs (both unlabelled and BODIPY-labelled). The graph shows the concentration on the Y-axis and size distribution on the X-axis. ( C ) Size-exclusion chromatography of unlabelled urinary small EVs revealed small EVs (peaks in the particle count graph) in fractions 6–8. ( D ) Exo-Check antibody array showing the presence of the following exosomal markers in the ultrafiltrated small EV urinary samples: FLOT1, ICAM, TSG101, CD81, and CD63. ( E ) Immunogold TEM (urinary small EVs) showing the presence of CD63 tetraspanin, an exosomal marker, in vesicles in the UF sample. ( F ) TEM of the ultrafiltrated medium collected from the CD63-GFP transduced human fibroblasts shows some structures displaying typical exosome features. Scale bar = 0.5 µm. ( G ) NTA of the genetically tagged fibroblast-derived small EVs: Analysis displays the light-scatter results (small EV mean size of 146 nm).

Article Snippet: The grids were incubated overnight with a mouse anti-human CD63 antibody (1:100, DSHB hybridoma H5C6, deposited by August, J.T./Hildreth, J.E.K.) as described in [ ].

Techniques: Isolation, Membrane, Concentration Assay, Size-exclusion Chromatography, Ab Array, Marker, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Isolation of Human Small Extracellular Vesicles and Tracking of Their Uptake by Retinal Pigment Epithelial Cells In Vitro

doi: 10.3390/ijms21113799

Figure Lengend Snippet: Uptake of urinary BODIPY-labelled small EVs and CD63-GFP tagged fibroblast-derived small EVs in ARPE19 cells. ( A ) Incubation of the ARPE-19 cells with BODIPY-labelled small EVs that were purified to remove unbound dye. ( B ) ARPE-19 cells exposed to purified (removal of small-sized vesicles) BODIPY solution (control (ctrl)) where no internalized fluorescence should be detected. ( C ) Uptake of fibroblast-derived CD63-GFP tagged small EVs in ARPE19 cells. ( D ) Higher magnification of C showing intracellular CD63-GFP expression that visualized tagged internalized small EVs.

Article Snippet: The grids were incubated overnight with a mouse anti-human CD63 antibody (1:100, DSHB hybridoma H5C6, deposited by August, J.T./Hildreth, J.E.K.) as described in [ ].

Techniques: Derivative Assay, Incubation, Purification, Control, Fluorescence, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Isolation of Human Small Extracellular Vesicles and Tracking of Their Uptake by Retinal Pigment Epithelial Cells In Vitro

doi: 10.3390/ijms21113799

Figure Lengend Snippet: Uptake of urinary BODIPY-labelled small EVs and CD63-GFP tagged fibroblast-derived small EVs in human iPSC derived RPE cells. ( A ) Incubation of iPSC-derived RPE cells with BODIPY-labelled small EVs. ( B ) iPSC-derived RPE cells exposed to CD63-GFP fluorescent tagged primary fibroblast-derived small EVs also showed intracellular exosome localization.

Article Snippet: The grids were incubated overnight with a mouse anti-human CD63 antibody (1:100, DSHB hybridoma H5C6, deposited by August, J.T./Hildreth, J.E.K.) as described in [ ].

Techniques: Derivative Assay, Incubation